Post-transplant lymphoproliferative disease (PTLD) is one of the most serious and potentially fatal post-transplant complications. Frequently, PTLD is triggered in the transplant recipient by the transfer of latent Epstein-Barr virus (EBV) present in the donor organ or tissue. The risk of PTLD is even higher when the donor is EBV seropositive and the recipient seronegative. Pervasiveness of EBV infection in the population is such that avoiding donor grafts infected with EBV is not a viable strategy. Current clinical practices of PTLD prevention requires patients to undergo 1) long term prophylactic antiviral regimens, 2) frequent monitoring via blood draws, and 3) adjustment of immunosuppression dosage, which can potentially lead to graft rejection. All these lead to high cost and high patient morbidity. There is also currently no antiviral drug that can treat the latent EBV infection because existing antiviral drugs only target proteins, and as such can only attack actively replicating virus. Overall, there is need to develop a more efficacious and specific way to prevent PTLD and its associated co-morbidities. The long term objective of this proposal is to eliminate the risk of donor-derived, EBV-associated PTLD for the ~50,000 annual tissue and organ transplant recipients in the US. It is hypothesized that a highly specific anti-viral therapeutic using CRISPR/Cas9 to directly target destruction of the EBV DNA genome would be able to eliminate the latent EBV reservoir in donor allografts. By treating the graft ex-vivo prior to transplantatio using this nuclease-based technology, the risk of donor EBV-derived PTLD is eliminated. Preliminary results demonstrate that this technology can successfully reduce viral load in patient-derived Burkitt's lymphoma cells with latent EBV infection, and can induce dramatic proliferation arrest reactivation of apoptotic pathways. These results demonstrate success of this technology in eliminating latent EBV virus from a cell line without observing any obvious cytotoxicity, thus this proposal aims to extend this work and evaluate the feasibility of this therapeutic approach in primary human blood samples in the context of hematopoietic cell transplants (HCT). This Phase I study will lay the foundation for future work to demonstrate efficacy and safety of this therapeutic in animal models and for solid organ transplant applications, and eventually for human trials. Specific Aim 1: Establish the efficacy of CRISPR/Cas9 anti-viral therapeutics in eliminating EBV infection from naturally infected EBV seropositive bone marrow samples, and evaluate associated effects on HSC function. Specific Aim 2: Investigate the safety of CRISPR/Cas9 anti-viral therapeutics in the context of eliminating EBV from EBV+ primary bone marrow samples, and optimize treatment conditions to minimize off-target effects on human host genome.